The polyamines, spermine and spermidine, are required for normal cell function and growth. Polyamine levels vary rapidly in response to the cell cycle. Levels are increased by biosynthesis and uptake, and decreased by catabolism and excretion. There is convincing evidence from clinical trials that the enzymes of the polyamine biosynthetic pathway are valid targets for the design of chemotherapeutic agents. In Project 2 we will carry out crystallographic studies of the polyamine biosynthetic enzymes S-adenosylmethionine decarboxylase (AdoMetDC), spermine synthase and spermidine synthase, and the polyamine catabolic enzyme, spermine/spermidine N1-acetyl transferase (SSAT). AdoMetDC is a key regulatory enzyme in polyamine biosynthesis. Its product, decarboxylated AdoMet, combines with putrescine to form spermidine or with spermidine to form spermine in reactions catalyzed by spermidine synthase and spermine synthase, respectively. SSAT catalyzes the rate-limiting step in polyamine catabolism. It is highly inducible and has a very short halflife. Cellular levels of SSAT are increased by compounds that prevent its degradation. Such compounds have been shown to be useful as chemotherapeutic agents either alone or in combination with inhibitors of polyamine biosynthesis. The enzymes for the crystallographic studies will be provided by Dr. Pegg in Project 1, who will also carry out biochemical studies. The crystallographic coordinates will be provided to Dr. Guida in Project 3 and Dr. Secrist in Project 4 for inhibitor design. We have already crystallized AdoMetDC and determined its structure alone and complexed with several reversible and irreversible inhibitors. We will determine the structures of AdoMetDC complexes and of AdoMetDC mutants as part of the overall drug design project. We have also crystallized SSAT, and over expressed and purified spermidine synthase and spermine synthase. We will initiate structural studies to further evaluate these potential targets.